Ceramide plays important roles in several aspects such as cell apoptosis, growth and differentiation, as well as signal transduction.Recently Ceramide is unveiled to be responsible for insulin resistance.Analysis of ceramides is challenging due to its low abundance.SPE (solid phase extraction) and mild alkalinehydrolysis are reported to be used to remove other lipids thus to enrich ceramides.Such methods are complicated and will lose information of other lipids.Here UHPLC-LTQ-Orbitrap was adopted for untargeted lipid profiling of cellular lipid extract.The UHPLC separation was performed on a Waters C8 column (1.7 μm, 2.1* 100 mm) by gradient elution, and data was acquired in ESI(+) in resolution 30,000 after ESI source parameters were optimized by tuning with ceramide standard.